ESIPT-based ratiometric fluorescence probe for the intracellular imaging of peroxynitrite

In this work, we set out to develop an endoplasmic reticulum (ER) directed ESIPT-based ONOO^- ratiometric fluorescent probe (ABAH-LW). ABAH-LW was synthesized in four steps and found to have a high sensitivity and selectivity towards the detection of ONOO^-. ABAH-LW was able to detect low concentrations of ONOO^- (limit of detection = 21.4 nM) within seconds producing a ratiometric change in fluorescence intensity. ABAH-LW further demonstrated the ability to ratiometrically image endogenous and exogenous ONOO^- in HeLa cells. Moreover, co-localization experiments were carried out using commercially available ER-Tracker Red, Lyso-Tracker Red and Mito-Tracker-Red, which were co-stained with ABAH-LW in HeLa cells. For ER-Tracker Red, Pearson's correlation co-efficient of 0.93 was determined and 3D surface plot analysis illustrated a large overlap between ABAH-LW and ER-Tracker Red using both red and blue channels. In addition some co-localisation with Mito-Tracker Red and ABAH-LW was observed (0.73).